Clustering

This notebook will begin looking at clustering methods on the expression of the genes in a single sample of the dataset of interest, from an unbiased approach.

Set Up

# Load libraries
library(magrittr)
library(scater)
library(readr)
library(bluster)
library(ggpubr)
library(pheatmap)

# Set file paths
data_dir <- file.path("results", "Gawad_processed_data")

# Source custom functions script
source(file.path("utils", "clustering-functions.R"))

Read in data

sample_290_normalized <- read_rds(
  file.path(data_dir, "SCPCS000216", "SCPCL000290_miQC_downstream_processed_normalized_reduced_sce.rds"))

Perform clustering

k-means

# Perform k-means clustering
sample_290_normalized <- kmeans_clustering(
  sample_290_normalized,
  params_range = c(4:10),
  step_size = 1,
  check_stability = TRUE
)

# grab column names with clustering results
kmeans_cols <- grep("kmeans", colnames(colData(sample_290_normalized)))
kmeans_cluster_names <- colnames(colData(sample_290_normalized)[, kmeans_cols])

# Plot k-means
kmeans_plot_list <- kmeans_cluster_names %>%
  purrr::map(~ plotReducedDim(sample_290_normalized, dimred = "UMAP", colour_by = .x) + 
               theme_bw()+
               theme(text = element_text(size = 22)))

cowplot::plot_grid(plotlist = kmeans_plot_list, ncol = 4)

graph-based, walktrap

# Perform graph-based walktrap clustering
sample_290_normalized <- graph_clustering(
  sample_290_normalized,
  params_range = c(5:25),
  step_size = 5,
  weighting_type = "rank",
  cluster_function = "walktrap",
  check_stability = TRUE
)

detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'

# grab column names with clustering results
walktrap_cols <- grep("walktrap", colnames(colData(sample_290_normalized)))
walktrap_cluster_names <- colnames(colData(sample_290_normalized)[, walktrap_cols])

# Plot
walktrap_plot_list <- walktrap_cluster_names %>%
   purrr::map(~ plotReducedDim(sample_290_normalized, dimred = "UMAP", colour_by = .x) + 
                theme_bw() +
                theme(text = element_text(size = 22)))

cowplot::plot_grid(plotlist = walktrap_plot_list, ncol = 3)

graph-based, louvain

# Perform graph-based louvain clustering
sample_290_normalized <- graph_clustering(
  sample_290_normalized,
  params_range = c(5:25),
  step_size = 5,
  weighting_type = "jaccard",
  cluster_function = "louvain",
  check_stability = TRUE
)

detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'detected tied distances to neighbors, see ?'BiocNeighbors-ties'

# grab column names with clustering results
louvain_cols <- grep("walktrap", colnames(colData(sample_290_normalized)))
louvain_cluster_names <- colnames(colData(sample_290_normalized)[, louvain_cols])

# Plot
louvain_plot_list <- louvain_cluster_names %>%
   purrr::map(~ plotReducedDim(sample_290_normalized, dimred = "UMAP", colour_by = .x) + 
                theme_bw() +
                theme(text = element_text(size = 22)))


cowplot::plot_grid(plotlist = louvain_plot_list, ncol = 3)

Check cluster validity stats

k-means

# Check the k-means cluster validity stats for each of the clusters and return
# stats in a data frame
kmeans_stats_df <- create_metadata_stats_df(sample_290_normalized, c(4:10), 1, "kmeans")

Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"

# Preview the results
head(kmeans_stats_df)

# Summarize the stats and return in a data frame
kmeans_summary_stats_df <- summarize_clustering_stats(kmeans_stats_df)

# Preview the summary results
head(kmeans_summary_stats_df)

purity plots

# Plot individual cluster purity stats
kmeans_purity_plots <- plot_cluster_purity(kmeans_stats_df)

kmeans_purity_plots

silhouette width plots

# Plot individual cluster silhouette width stats
kmeans_silhouette_plots <- plot_cluster_silhouette_width(kmeans_stats_df)

kmeans_silhouette_plots

graph-based, walktrap

# Check the walktrap cluster validity stats for each of the clusters and return
# stats in a data frame
walktrap_stats_df <- create_metadata_stats_df(sample_290_normalized, c(5:25), 5, "walktrap")

Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"

  
# Preview the all stats results
head(walktrap_stats_df)

# Summarize the stats and return in a data frame
walktrap_summary_stats_df <- summarize_clustering_stats(walktrap_stats_df)

# Preview the summary results
head(walktrap_summary_stats_df)

purity plots

# Plot individual cluster purity stats
walktrap_purity_plots <- plot_cluster_purity(walktrap_stats_df)

walktrap_purity_plots

silhouette width plots

# Plot individual cluster silhouette width stats
walktrap_silhouette_plots <- plot_cluster_silhouette_width(walktrap_stats_df)

walktrap_silhouette_plots

graph-based, louvain

# Check the louvain cluster validity stats for each of the clusters and return
# stats in a data frame
louvain_stats_df <- create_metadata_stats_df(sample_290_normalized, c(5:25), 5, "louvain")

Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"
Joining, by = "cell_barcode"

# Preview the results
head(louvain_stats_df)

# Summarize the stats and return in a data frame
louvain_summary_stats_df <- summarize_clustering_stats(louvain_stats_df)

# Preview the summary results
head(louvain_summary_stats_df)

purity plots

# Plot individual cluster purity stats
louvain_purity_plots <- plot_cluster_purity(louvain_stats_df)

louvain_purity_plots

silhouette width plots

# Plot individual cluster silhouette width stats
louvain_silhouette_plots <- plot_cluster_silhouette_width(louvain_stats_df)

louvain_silhouette_plots

Check cluster stability

k-means

# Check and plot cluster stability
kmeans_cluster_names %>%
  purrr::walk(~ plot_cluster_stability(sample_290_normalized, .x))

graph-based, walktrap

walktrap_cluster_names %>%
  purrr::walk(~ plot_cluster_stability(sample_290_normalized, .x))

graph-based, louvain

louvain_cluster_names %>%
  purrr::walk(~ plot_cluster_stability(sample_290_normalized, .x))

Session info

sessionInfo()

R version 4.1.1 (2021-08-10)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Catalina 10.15.7

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats4    stats     graphics  grDevices datasets  utils     methods   base     

other attached packages:
 [1] pheatmap_1.0.12             ggpubr_0.4.0                bluster_1.4.0               readr_2.1.1                 scater_1.22.0              
 [6] ggplot2_3.3.5               scuttle_1.4.0               SingleCellExperiment_1.16.0 SummarizedExperiment_1.24.0 Biobase_2.54.0             
[11] GenomicRanges_1.46.1        GenomeInfoDb_1.30.0         IRanges_2.28.0              S4Vectors_0.32.3            BiocGenerics_0.40.0        
[16] MatrixGenerics_1.6.0        matrixStats_0.61.0          magrittr_2.0.1             

loaded via a namespace (and not attached):
 [1] bitops_1.0-7              RColorBrewer_1.1-2        rprojroot_2.0.2           tools_4.1.1               backports_1.4.0           utf8_1.2.2               
 [7] R6_2.5.1                  irlba_2.3.3               vipor_0.4.5               colorspace_2.0-2          nnet_7.3-16               withr_2.4.3              
[13] tidyselect_1.1.1          gridExtra_2.3             compiler_4.1.1            cli_3.1.0                 BiocNeighbors_1.12.0      DelayedArray_0.20.0      
[19] labeling_0.4.2            scales_1.1.1              stringr_1.4.0             digest_0.6.29             rmarkdown_2.11            XVector_0.34.0           
[25] pkgconfig_2.0.3           htmltools_0.5.2           sparseMatrixStats_1.6.0   fastmap_1.1.0             rlang_0.4.12              rstudioapi_0.13          
[31] DelayedMatrixStats_1.16.0 jquerylib_0.1.4           farver_2.1.0              generics_0.1.1            jsonlite_1.7.2            BiocParallel_1.28.2      
[37] car_3.0-12                dplyr_1.0.7               RCurl_1.98-1.5            BiocSingular_1.10.0       modeltools_0.2-23         GenomeInfoDbData_1.2.7   
[43] Matrix_1.3-4              Rcpp_1.0.7                ggbeeswarm_0.6.0          munsell_0.5.0             fansi_0.5.0               abind_1.4-5              
[49] viridis_0.6.2             lifecycle_1.0.1           stringi_1.7.6             yaml_2.2.1                carData_3.0-4             zlibbioc_1.40.0          
[55] flexmix_2.3-17            grid_4.1.1                parallel_4.1.1            ggrepel_0.9.1             crayon_1.4.2              lattice_0.20-45          
[61] splines_4.1.1             cowplot_1.1.1             beachmat_2.10.0           hms_1.1.1                 knitr_1.36                pillar_1.6.4             
[67] igraph_1.2.9              optparse_1.7.1            ggsignif_0.6.3            ScaledMatrix_1.2.0        glue_1.5.1                evaluate_0.14            
[73] renv_0.14.0               BiocManager_1.30.16       vctrs_0.3.8               tzdb_0.2.0                gtable_0.3.0              getopt_1.20.3            
[79] purrr_0.3.4               tidyr_1.1.4               xfun_0.28                 rsvd_1.0.5                broom_0.7.10              miQC_1.2.0               
[85] rstatix_0.7.0             viridisLite_0.4.0         tibble_3.1.6              tinytex_0.35              beeswarm_0.4.0            cluster_2.1.2            
[91] ellipsis_0.3.2            here_1.0.1